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J Biol Chem, Vol. 274, Issue 36, 25921-25926, September 3, 1999
From the Ornithine decarboxylase (ODC) declines in cells
that accumulate an excess of polyamines, the downstream products of the
enzyme. Superfluous production of polyamines is thus prevented. In
animal cells, polyamines reduce ODC activity by accelerating its
degradation. Similar down-regulation of ODC activity has been observed
in the budding yeast Saccharomyces cerevisiae, but induced
degradation has not been documented. Here we show using pulse-chase
analysis that the loss of enzyme activity is the result of increased
degradation of ODC. Polyamines reduce the half-life of the newly
synthesized protein from 3 h to approximately 10 min. Degradation
of bulk ODC pools is also accelerated by polyamines, but the absolute rate of turnover is slower, with a half-life of 5 h in untreated and 1 h in treated cells. Newly synthesized ODC polypeptide thus undergoes a process of maturation that renders it relatively resistant to both basal and polyamine-induced degradation. Proteasome mutants have a blunted or absent regulatory response, implicating both the core
protease and the regulatory cap of the proteasome in induced
degradation of yeast ODC.
The enzymes that control polyamine metabolism share a common
characteristic: the regulation of ornithine decarboxylase
(ODC)1 (1, 2),
S-adenosylmethionine decarboxylase (3), and spermidine/spermine acetyltransferase (4) depends in large part on
post-translational mechanisms. This property has perhaps been
documented most fully for ODC of animal cells (1, 2). ODC catalyzes the
initial step in the biosynthesis of polyamines, small, ubiquitous,
abundant, and essential cellular polycations (5). When these rise to
excess levels, the half-life of the enzyme becomes shorter, and its
steady-state level falls. This sequence of events provides a form of
feedback regulation whereby the end products of the biosynthetic
pathway limit the activity of the initial enzyme of the pathway. A
second protein, termed antizyme, controls the process. Production of
antizyme requires a translational frameshift to align a small upstream
ORF with a second ORF that encodes all known functions of the protein. Polyamines greatly enhance frameshifting, and thus control the level of
antizyme (6). Antizyme associates with ODC, disrupting the homodimeric
enzyme and rendering it inactive. The ODC:antizyme heterodimer thus
formed is degraded by the proteasome (7). ODC represents an unusual
substrate for the proteolytic action of the proteasome. Proteasomes act
predominantly on proteins that have been modified by covalent
association with multiple copies of the protein ubiquitin (8), but
accelerated ODC degradation depends instead on 1:1 stoichiometric
noncovalent association with antizyme (9, 10).
In the budding yeast Saccharomyces cerevisiae, the mechanism
of regulation of ODC (yODC) is less well understood. However, many key
attributes are similar to those observed in animal cells (11, 12).
Augmenting polyamines reduces yODC activity and protein, without
changing the amount of yODC mRNA; changes in transcription or yODC
mRNA stability are therefore implausible as mechanisms of control.
Polyamines do not change the distribution of yODC mRNA on the
polyribosomes, implying that translation is unaltered. Expression of
the yODC open reading frame without flanking regions of the mRNA
confers activity that remains under the control of polyamines,
suggesting that the protein itself is subject to control (11).
Degradation of yODC in response to polyamines subsists as the most
probable explanation both because it is not excluded by the data and
because animal cells utilize this mechanism. Direct evidence for this
conclusion has, however, proven elusive. In yeast, genetic evidence has
demonstrated that the proteasome digests yODC (13, 14), but polyamine
excess has not been shown to influence this process. Here we provide
evidence that polyamines accelerate the degradation of yODC by the
proteasome in yeast.
Culture of Yeast--
Cells were grown in liquid culture with
aeration by shaking at 30 or at 25 oC for proteasome
mutant strains. Synthetic minimal medium (SM) consisted of 0.67% yeast
nitrogen base (Difco), 2% glucose, and amino acid omission mixtures
(BIO 101, Inc.), deficient in the appropriate amino acids required for
selection. Transformation of yeast was done by the lithium acetate
method (15). Polyamine treatment was with 1 mM spermidine,
1 mM spermine. Yeast manipulations were carried out as
described (16). General methods for DNA manipulation made use of
standard procedures (17).
Strains and Plasmids--
The wild type background strain used
in these studies was PSY93 (mat yODC Enzymatic Activity--
Cells were washed in
H2O and resuspended in 450 µl of lysis buffer (0.02%
Brij, 25 mM Tris pH 8.0, 0.1 mM EDTA, 2 mM dithiothreitol, 0.1 mM pyridoxal phosphate,
0.1% Triton X-100, 1 mM Mg Cl2), disrupted by
agitation with glass beads (BioSpec Bead-Beater), and the 14,000 × g supernatant recovered for determination of protein
concentration (Bradford, Bio-Rad, bovine serum albumin standard) and
yODC enzymatic activity, using an assay that measures release of
14CO2 from 14C-carboxy-labeled
ornithine (12). Enzymatic activity units are expressed as pmol of
CO2 released/min/mg of protein. A protease inhibitor mix
containing phenylmethylsulfonyl fluoride, leupeptin, aprotinin, and
pepstatin A (Roche Molecular Biochemicals) was included in the lysis buffer.
Metabolic Labeling of Cells and yODC
Immunoprecipitation--
Cultures in mid-logarithmic phase
(OD600 0.2 to 0.5) in SM medium at 30o were
washed once in labeling medium (identical to SM, but without methionine
and equimolar NH4Cl replacing
(NH4)2SO4), resuspended in the same
medium to a final cell density of 2 ODU600, cultured for
30-40 min, and then labeled for 3 min by addition of S35
Express label (ICN) containing [35S]methionine and
35S-cysteine. When cells were labeled for longer periods,
cold methionine and cysteine were added with S35 Express
label to prolong incorporation; acid-precipitable incorporation of
35S was found to be linear for at least 3 h under
these conditions. Chase was initiated by centrifuging the cells and
resuspending them in pre-warmed SM containing unlabeled 1 mM cysteine and 1 mM methionine. Aliquots were
collected periodically and diluted into ice-cold tubes containing
NaN3, 30 mM final concentration. Cells were
centrifuged, washed and stored at Colony and Western Immunoblot Assay of yODC--
An assay
developed to measure yeast carboxypeptidase Y (24) was adapted to
assess the level of yODC in cells grown on nitrocellulose filter
overlays in SM agar plates. Yeast strains were streaked onto
nitrocellulose filters (Hybond-C, Amersham Pharmacia Biotech) placed
upon agar plates containing the SM medium. Polyamines, where indicated,
were added to the medium subsequent to plate preparation and before
application of filters. The cells were allowed to grow for 3 days at
25 oC and then lysed upon the filters (24). Filters were
developed using the Amersham ECL detection kit and protocol. Filters
were blocked in TBS-T including 5% powdered low-fat milk and 5%
bovine serum albumin. Primary anti-yODC antibody was identical to that used for immunoprecipitation (1:10,000 dilution) in TBS-T plus 0.3%
bovine serum albumin. Secondary antibody was goat anti-rabbit conjugated with horseradish peroxidase (Sigma, 1:15,000 dilution) in
TBS-T. For Western blot analysis, cells were treated with polyamines or
were untreated, extracts prepared via glass bead lysis, subjected to
SDS-PAGE, and immunoreactive yODC visualized by ECL as above. Equality
of sample loading was verified by using monoclonal antibody YL1/2 (25)
directed against yeast tubulin.
Response of yODC Activity and Protein to Polyamines--
Polyamine
excess causes a reduction of ODC activity in yeast and other
eukaryotes. We created a yeast strain with two kinds of genetic changes
intended to make investigation of this form of regulation more
tractable. First, a null mutation was introduced in SPE2.
Its gene product, S-adenosylmethionine decarboxylase, is
required for conversion of putrescine, the direct product of yODC, to
polyamines (18). By thus preventing endogenous synthesis of polyamines,
cellular pools could be made to depend solely on the amounts of
polyamines provided in the medium. Because polyamines are essential,
spermidine and spermine were added to the medium at a concentration of
10
To determine whether polyamines reduce yODC protein as well as
activity, we used a colony-based immunologic assay (24). Cells were
transferred to nitrocellulose filters, and grown as an overlay on
synthetic complete medium plates supplemented or not with polyamines.
After cell lysis, the amount of immunoreactive yODC present on the
filters was assessed. We tested the 3x strain, as well as a strain in
which the yODC gene had been disrupted, the latter to determine the
intensity of assay background. Incubation with polyamines for 3 days, a
period required to establish grossly visible colonies, significantly
reduced the amount of yODC detected in the 3x strain (Fig.
1B). To confirm this inference and to determine whether
regulation of protein level takes place more promptly, we used Western
immunoblot analysis of extracts of cells exposed to polyamines in
suspension culture for only 4 h. Immunoreactive yODC declined
markedly as a result of this treatment (Fig. 1C). No
immunoreactive yODC was detected in the spe1 disruption
strain. Because polyamines induced a comparable reduction of yODC
activity and yODC protein within hours, we conclude that the loss of
activity results from a diminution of protein level.
Degradation of yODC--
To test directly whether polyamine
treatment of cells accelerates yODC degradation, we metabolically
labeled cells and measured how fast labeled yODC diminished in cells
that were pretreated with polyamines or were untreated. The labeling
time was 3 min (pulse), followed by subsequent incubation under
conditions that prevented further labeling of newly synthesized
proteins (chase). Extracts were immunoprecipitated with antibody
specific for yODC, and labeled immunoprecipitated protein was
visualized by SDS-PAGE and phosphoimager processing. The specificity of
precipitation was verified using yODC gene disruption and overexpressor
strains (Fig. 2A). yODC in 3x
strain cells not pretreated with polyamines had a half-life of about
3 h (Fig. 2B). Polyamine pretreatment reduced the
half-life to about 10 min. Although signal intensity declined much
faster in the treated cells, the intensity of yODC labeling at the
earliest time point observed was not strongly or consistently
influenced by polyamine treatment. These results imply that polyamine
treatment accelerates degradation more than ten-fold, but has little or
no effect on synthesis. Pulse-chase experiments with 1x cells produced
results that were qualitatively similar to those seen with the 3x
strain (results not shown), but signal strength was diminished, making
data analysis problematic. Consistent with the observation that
activity is not regulated in a strain expressing yODC from a high copy
plasmid (11),2 pulse-chase
experiments showed that yODC has about the same 3-h half-life as in the
3x strain and that polyamines do not stimulate turnover (Fig.
2C).
Degradation of Steady-state yODC Pools--
The pulse-chase
experiments described above demonstrate that polyamine treatment
labilizes newly synthesized yODC protein, those molecules metabolically
labeled during the brief 3-min pulse employed. Pools of yODC that exist
prior to treatment may be made similarly labile. However, it is
possible instead that yODC undergoes a process of folding or other form
of maturation that renders it more stable. In that case, polyamines
might induce cells to accelerate the degradation only of newly
synthesized yODC, whereas pre-existing pools of the molecule decay with
the 3 h half-life characteristic of newly synthesized yODC in
untreated cells or even remain entirely stable, decaying only through a
dilution process dependent on cell growth. To test this, cells were
labeled for 2.5 h before imposing polyamine treatment. Chase was
initiated simultaneously with polyamine treatment, and the amount of
labeled yODC was assessed during a subsequent 4-h chase period (Fig.
3). As was true for yODC populations
labeled for 3 min, polyamine treatment also accelerated the degradation
of long labeled yODC. However, the absolute rate of degradation after
polyamine treatment is much slower in the second case. In contrast to
short-labeled yODC, for which polyamines changed the half-life from
3 h to 10 min, polyamines altered the half-life of long-labeled
yODC from 5 h to 60 min. In summary, in cells with either a basal
or augmented level of polyamines, yODC undergoes a process of
maturation that reduces its susceptibility to degradation.
Effect of Proteasome Mutations--
Both mammalian ODC and yeast
ODC are degraded in vitro by the yeast proteasome. In
vivo, mutations in the yeast proteasome impede degradation of
yODC, but these experiments have been carried out in cells not subject
to treatment with polyamines. To determine whether the accelerated form
of degradation induced by polyamines also depends on the proteasome, we
utilized two temperature-sensitive mutants: the first, a double mutant
(pre1-1,pre2-2) which impairs four of the six proteolytic
sites of the 20 S proteasome core (19); the second
(cim3-1), a temperature-sensitive mutant of the Rpt6 ATPase
subunit of the regulatory cap of the 26 S proteasome (20). In
pre1-1,pre2-2 cells, polyamines produced a 2-fold
reduction, compared with a 28-fold change in the corresponding wild
type cells (Table I). In
cim3-1 cells, the polyamine-induced change was 0.5-fold (a
small increase rather than reduction), compared with 8-fold for the
wild type. Mutations of either the core proteolytic chamber or
regulatory complex interfere with polyamine-induced reduction of yODC
activity, presumably by interfering with polyamine-induced accelerated
degradation.
As described in "Introduction," previous findings suggested
that cellular polyamines reduce yODC activity and protein by
accelerating the degradation of the enzyme. However, the low level of
expression of yODC has impeded direct measurements of stability. We
constructed well regulated cells with augmented yODC expression and
produced highly specific antiserum ("Experimental Procedures").
These technical tools made it possible to measure changes in the level
of immunoreactive yODC and to perform pulse-chase analysis to measure
its rate of turnover. We found that polyamine treatment greatly reduces
the amount of yODC and diminishes the half-life of newly synthesized yODC, reducing it from about 3 h to 10 min. These changes in
degradation were not associated with altered synthesis; using a short
pulse labeling time of 3 min, polyamines were found to cause no
consistent alteration in metabolic labeling of yODC. This result is
consistent with that previously found in animal cells (26). Although it is difficult to exclude small changes in translation rate, it is clear
that the bulk and perhaps all of the polyamine-induced change in yODC
activity results from a post-translational process. This is in contrast
to the fungus Neurospora crassa, in which polyamines exert a
repressive influence on ODC by reducing the abundance of ODC mRNA
(27).
Previous investigations of yODC stability employed inhibitors of
synthesis, predominantly cycloheximide, to assess the rate at which
activity falls when synthesis of proteins is halted (11, 12). These
studies showed that by this measure of stability, yODC has a half-life
of more than an hour, regardless of whether or not polyamines are
augmented. These results are only apparently discrepant with those
reported here. Two models could reconcile these results with ours.
Model 1 asserts that yODC is subject to accelerated degradation only or
predominantly during a brief period after translation, perhaps before
the newly synthesized polypeptide folds into an enzymatically active
conformation. In that case, steady-state pools of enzymatically active
yODC, those that are determined in cycloheximide chase experiments,
would be predominantly in a matured form no longer susceptible to rapid degradation. Experiments using inhibitors of synthesis would reveal little or no effect of polyamines on degradation. To directly test
this, we carried out prolonged labeling to uniformly label yODC pools.
Cells were labeled for 2.5 h before initiating a chase. At the end
of the label period, most of the labeled yODC cohort present will have
been synthesized more than an hour ago, a calculation based on an 3-h
yODC half-life and a 2-h cell division time. These labeled yODC pools
were then chased; at the time of initiation of the chase, cells were
either treated with polyamines or left untreated. In long labeled cells
not treated with polyamines, labeled yODC pools declined with a 5-h
half-life. In polyamine-treated cells, the half-life was reduced to
1 h. Polyamines therefore accelerate degradation of long-labeled
yODC 5-fold. These results show that model 1 is untenable.
Model 2 offers a offers a different resolution of the apparent conflict
between cycloheximide chase versus pulse-chase experiments: simultaneous treatment with polyamines and inhibitors of protein synthesis may prevent the production of a polyamine-induced protein required for accelerated degradation. This is the case in animal cells.
There the more marked effect on ODC activity of treatment with
polyamines alone, compared with cycloheximide or cycloheximide plus
polyamines (28), is because of the polyamine-induced synthesis of the
protein antizyme, which binds to ODC and accelerates its degradation.
Cycloheximide prevents antizyme synthesis but allows pre-formed ODC to
be degraded at a rate characteristic of an antizyme-independent process
or one dependent on the lesser amount of antizyme present in cells with
basal levels of polyamines. In yeast as well, cycloheximide plus
polyamines produce changes in yODC activity identical to those seen
with cycloheximide and slower than those seen with polyamines alone
(12). This suggests that, in yeast too, excess polyamines stimulate the
synthesis of a protein responsible for degradation. In yeast, polyamine
treatment produces similar kinetics of decline in yODC activity,
regardless of whether or not cycloheximide treatment is superimposed an
hour after polyamines are added (12). This too is consistent with the
possibility that polyamines cause a protein to appear which accelerates
degradation of yODC and which can persist if synthesis is later
impeded. Model 2 is fully compatible with our data and that of others.
Our attempts to obtain direct biochemical evidence for induction in
yeast of a hypothetical functional analog of antizyme have, however,
been unsuccessful. Such an analog is likely to act catalytically on its
substrate, as does mammalian antizyme, and hence be present in cells in
a still smaller amount than the already scarce yODC. This suggests that
genetic approaches to its identification will likely prove more useful
than attempts at biochemical isolation. Whatever the mechanism of yODC
regulation, amounts greatly in excess of that normally present in the
cell exceed its capacity (Ref. 11 and Fig. 2C). As shown
here, a modest degree of over-expression, about 3-fold normal, is
compatible with full or near-full regulation. The nature of the
component limiting regulatory capacity remains to be determined, as
does its relationship with the hypothetical yeast antizyme analog.
Proteasome mutations in either the catalytic 20 S core (29) or 19 S
regulatory cap (13) interfere with yODC degradation in cells with basal
polyamine levels. The same mutations are here shown to strongly impede
polyamine-induced reduction in yODC. Induced degradation as well as
basal degradation therefore depends on the integrity of both these
functional elements of the proteasome, the core, and regulatory cap. As
both elements of the yeast proteasome are required in vitro
for proteolysis of mouse and yeast ODC (14), it is very likely that the
proteasome itself is the agent of induced yODC degradation.
The observed difference in polyamine-induced lability of "new"
versus "aged" yODC is not because of the use of a 4-h
polyamine pre-treatment before pulse-chase for the short label
experiment, versus addition of polyamines at initiation of
the chase in the long label experiment. A treatment period of 1 h
is in fact sufficient to fully establish yODC lability. That 1 h
suffices to establish lability is consistent with our data and that of
others (Ref. 12, and Fig. 1A) and directly supported by our
observation that the short label experiment yields an identical result
if a 1-h rather than 4-h pretreatment is used (data not shown). Hence
the cellular conditions required to produce extreme lability of newly synthesized yODC had been established within one h. of inception of the
chase in the long label experiment. At what point after synthesis does
yODC become less susceptible to degradation? Compared with newly
synthesized yODC, "mature" molecules degrade about 2-fold more
slowly in untreated cells (half-life 3 h versus 5 h) and about 6-fold more slowly in polyamine-treated cells (half-life 10 min versus 60 min). We know little about the time scale
over which maturation takes place because we compared cohorts of
molecules labeled for very dissimilar times, 3 min or 2.5 h. The
timing and mechanism of the transition of yODC from more to less
degradation-susceptible, therefore, remain highly conjectural. The
relevant transition could be associated with one or more of several
steps: completion of polypeptide translation, folding to a native
monomeric configuration, self-association of monomers to form the
enzymatically active homodimer, or association with some other cellular
component. Two decades ago, Wheatley et al. (30)
demonstrated using pulse-chase labeling of cultured mammalian cells
that newly synthesized proteins are surprisingly labile. After a 5-min
pulse with 3H-leucine, about one third of incorporated
(acid precipitable) counts were hydrolyzed by cells within 1 h, a
fraction that fell progressively as the labeling time was increased.
This result was interpreted to indicate that a large class of nascent
proteins are initially at high risk for degradation and then undergo
time-dependent stabilization. It is possible that yODC
represents but a special case of this general phenomenon.
Is accelerated degradation sufficient to account for the reduction in
yODC activity observed in cells exposed to high polyamine concentrations? This treatment causes activity to fall with a 1-h
half-life (Ref. 12, and Fig. 1A). Some of the reduction in
activity is because of very fast degradation of newly synthesized yODC,
functionally equivalent to a near cessation of synthesis. The remaining
reduction in activity is the result of a change in the half-life of the
bulk steady-state yODC pool from 5 h to 1 h, a new rate
consistent with the rate of loss of enzymatic activity. Significantly,
polyamines cause similar kinetics of decline in enzymatic activity and
in long labeled yODC, implying that these are measuring different
properties of a common molecular population. Degradation therefore
accounts for loss of activity.
We thank Sudarsi Desta for technical assistance.
*
This work was supported by Public Health Service Grant
GM45335 from the NIGMS, National Institutes of Health.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed. Tel.:
415-476-1783; Fax: 415-476-8201; E-mail: pcoffin@itsa.ucsf.edu.
2
C. Toth and P. Coffino, unpublished results.
The abbreviations used are:
ODC, ornithine
decarboxylase;
ORF, open translational reading frame;
yODC, yeast
ornithine decarboxylase;
PAGE, polyacrylamide gel electrophoresis;
TBS-T, Tris-buffered saline with Tween 20.
Regulated Degradation of Yeast Ornithine Decarboxylase*
and§¶
Department of Microbiology and Immunology
and the § Department of Medicine, University of California,
San Francisco, San Francisco, California 94143-0414
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,leu2,ura3,trp1,his3) from
A. Johnson, University of California, San Francisco. In the
PSY93-derived strains described here, unless otherwise stated, the
SPE2 gene encoding S-adenosylmethionine decarboxylase was replaced by a spe2::LEU2
disruption using a plasmid obtained from C. Tabor and H. Tabor,
National Institutes of Health (18). The effect of this mutation was to
prevent conversion of putrescine to the polyamines spermidine and
spermine. Proteasome mutant strains were pre1-1, pre2-2 in
a WCG4 strain background (19), or cim3-1 (20) backcrossed
4x to the W303 strain. Activity of yODC in each of these proteasome
mutant strains was compared with that in their respective isogenic or
congenic wild type control strain. The yODC (SPE1) genomic
clone (21) was obtained from W. Fonzi, University of California,
Irvine. A strain with three yODC gene copies (3x strain) was
constructed by cloning SPE1 expressed from its native
promoter in the CEN-ARS plasmids (22) pRS313 (HIS3) and pRS314 (TRP1), transforming
SPE1 cells with both constructs and subsequently maintaining
continuous selection for both markers. Cells with a single chromosomal
copy of SPE1 are termed 1x and isogenic cells with three
gene copies 3x.
80o for subsequent
analysis by immunoprecipitation. For immunoprecipitation of labeled
yODC, all manipulations were done at 4o, and all solutions
contained 1× protease inhibitors (Roche Molecular Biochemicals) and 1 mM phenylmethylsulfonyl fluoride in addition to the
indicated constituents. Cells were broken by glass bead agitation
(BioSpec Bead-Beater) in 200 µl of phosphate-buffered saline + 1%
Triton X-100 (PBS/TX-100). After lysis, 800 µl of PBS/TX-100 + 0.2%
SDS (wash buffer) was added, and the glass beads were briefly vortexed.
The solution was cleared by centrifugation at 14,000 × g. A portion of the high speed supernatant was used to
determine acid precipitable 35S, and the volumes used for
immunoprecipitation were adjusted to contain equal counts. Extracts
were pre-cleared by incubation with pre-immune serum and protein
A-Sepharose (Amersham Pharmacia Biotech), and yODC was then
immunoprecipitated with polyclonal rabbit antiserum prepared (Animal
Pharm Services) by immunizing rabbits with purified recombinant yODC,
produced in Escherichia coli. The anti-yODC antibody was
rendered monospecific before use by adsorption with an acetone powder
extract of yeast (23) in which the SPE1 gene was disrupted.
Antibody-yODC complexes were collected by addition of protein
A-Sepharose and washed sequentially in wash buffer, urea/NaCl buffer (2 M urea, 10 mM Tris, pH 7.5, 1% Triton X-100, 2 M NaCl), wash buffer, and low salt buffer (10 mM Tris, pH 7.5, 10 mM NaCl). The precipitates
were analyzed by SDS-PAGE, and radiolabeled bands were visualized by
autoradiography or by using a phosphoimager (Molecular Dynamics). The
density of bands corresponding to labeled yODC was measured by
ImageQuant (MD).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
7 each for routine cell propagation and added at a
level 10,000-fold higher to down-regulate yODC. Second, because yODC is
a low abundance protein, biochemical and molecular investigation of the
mechanism of the response would be facilitated by expressing it to a
higher level than that found in wild type cells. However, previous
investigations (11) have shown and we have confirmed that high level
expression of yODC from a multi-copy plasmid greatly blunts the
regulatory response. Therefore, the extent of overexpression must be
limited if the desired strain is to maintain regulation. To this end, we established a strain with three copies of the yODC gene (3x strain),
one chromosomal and two on plasmids, and measured the response. The
cells were treated with polyamines to expand intracellular pools, and
time-dependent changes in yODC activity were measured. As
has been previously reported for cells with a single gene copy, yODC
activity fell with a half-life of about 1 h, declining more than
50-fold within 8 h (Fig.
1A). The decline in activity
displayed similar kinetics to that observed in isogenic cells with a
single chromosomal copy of the yODC gene (1x strain).
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Fig. 1.
Loss of yeast ODC upon polyamine treatment in
cells with one or three gene copies. A,
time-dependent change in yODC activity. At the initial time
point, spermidine and spermine at concentrations of 1 mM
each were added to the medium of 1x ( 
)and 3x (
) strains. Portions
were removed periodically for measurement of yODC activity. The data
are plotted as a percent of initial activity, which was 27.8 and 60.8 pmol/min/mg of protein in the 1x and in the 3x strains, respectively.
B, immunoreactive yeast ODC in cell colonies after polyamine
treatment. Cells with three copies of the yODC gene (SPE1
3x) were patched to filters, treated with polyamines or left
untreated, and processed to display immunoreactive yODC. Cells with the
yODC gene disrupted (spe1::His3) were
processed in parallel and serve as a control for nonspecific
immunoreactivity. C, Western immunoblot analysis of yODC in
cell extracts after polyamine treatment. Cells with the yODC gene
disrupted (spe1::His3) or with three
copies of the yODC gene (SPE1 3x) were untreated or treated
with polyamines, as indicated, and yODC in extracts were visualized by
Western blotting. Immunoreactive bands were visualized with antibody to
yODC or to yeast tubulin, which serves as a loading control, as
indicated.
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Fig. 2.
Pulse-chase experiment to determine yODC
turnover. A, the specificity of yODC
immunoprecipitation from metabolically labeled cell extracts was
determined by comparing the pattern of labeled bands obtained using
cells with no wild type yODC gene copy ( ), with one copy
(1X), and with multiple copies carried on a 2 µM plasmid (HIGH COPY). The arrow
indicates the position of migration of yODC (52.3 kDa expected
molecular mass (21)) on the SDS-PAGE gel. B, cells with
three yODC gene (SPE1) copies and a spe2
disruption were treated for 4 h with spermidine and spermine at
concentrations of 1 mM each or were untreated. Each culture
was then pulse-labeled with [35S]methionine and
35S-cysteine for 3 min. Cells were transferred to medium
without label, and samples were removed periodically and processed for
determination of radiolabeled yODC. Chase times were 0, 3, 6, and 12 min for treated cells and 0, 30, 60, or 120 min for untreated cells.
The time-dependent changes in intensity of the radiolabeled
yODC protein of untreated () or treated () cells are plotted as a
percent of initial value. C, cells with multiple yODC gene
(SPE1) copies carried on a 2 µM plasmid and
wild type for SPE2 were treated, labeled, and analyzed as in
panel B. , untreated cells; , treated cells. Chase
times were 0, 30, 90, and 180 min.
View larger version (47K):
[in a new window]
Fig. 3.
Chase experiment to determine turnover of
radiolabeled yODC in cells subjected to a prolonged label period.
A 3x strain culture was metabolically labeled for 2.5 h. One portion of
the culture was chased in medium containing spermidine and spermine at
concentrations of 1 mM each and a second portion in medium
without polyamines. Chase samples were removed after 0, 2, and 4 h
and processed and analyzed as in Fig. 2B. , untreated
cells; , treated cells.
Comparison of yODC activities
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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